Pseudouridine is the most common post-transcriptional modification of RNA, and arises from the isomerization of uridine catalyzed by the pseudouridine synthases. These enzymes fall into four families that share no global sequence similarity but appear homologous on the basis of their tertiary structures. Several pseudouridine synthases are physiologically critical in prokaryotes and eukaryotes, including the human enzyme dyskerin, the absence of which causes the X-linked disease dyskeratosis congenita. Pseudouridine residues in small nuclear RNA are required for pre-mRNA splicing, an essential function in eukaryotes. Since the families of pseudouridine synthases are so highly divergent, it remains possible that they proceed by different mechanisms, and such differences may eventually be exploited in new classes of antibiotics or provide a better understanding of the disease dyskeratosis congenita. The proposed experiments will elucidate the chemical mechanism followed by two E. coli pseudouridine synthases of different families, RluA and TruB, which are particularly tractable targets for mechanistic work because they handle small RNA substrates (stem-loop oligonucleotides). RNA containing 5-fluorouridine has been used as an inhibitor and mechanistic probe of the pseudouridine synthases, favoring a mechanism involving a Michael addition. Recent results, however, indicate that TruB is not inhibited by 5-fluorouridine in RNA and that an assumed hydrolysis of a proposed intermediate does not occur, leading to doubt concerning the mechanistic conclusions based on earlier studies with the same compound. The proposed experiments will resolve issue concerning 5-fluorouridine in RNA and allow further mechanistic insight. Such insight will also be gained using RNA containing uridine with a 2'-fluoro group, similar to the series of remarkably informative experiments using fluorosugars to probe the mechanism of glycosidases. A combination of NMR, mass spectrometry, site-directed mutagenesis and kinetic analysis supplemented by X-ray crystallography will be employed to examine the effects of the two types of fluorinated RNA as well as site-directed mutagenesis of amino acid residues that appear to play a role in catalysis. Finally, synthesized small RNA substrates will be used to examine the substrate specificity of RluA.